A monoclonal antibody against four serotypes of dengue virus and its application

Abstract

The present invention provides a monoclonal antibody against four serotypes of dengue virus and its application, belonging to the technical field of monoclonal antibodies. The monoclonal antibody against four serotypes of dengue virus provided by the present invention has an amino acid sequence of the heavy chain as shown in SEQ ID NO.1, and an amino acid sequence of the light chain as shown in SEQ ID NO.2. The monoclonal antibody of the present invention has a strong binding ability with four serotypes of DENV virus, can detect four serotypes of dengue virus at the same time, and the antibody titer can reach 1:512000 or more, which has great application value.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibodies, and in particular to a monoclonal antibody against four serotypes of dengue viruses and applications thereof. Background Art

[0002] Dengue fever is an acute systemic infectious disease caused by dengue virus (DENV) transmitted by mosquitoes. It belongs to the genus Flaviviridae and is a single-stranded positive-strand RNA virus. The virus is resistant to low temperatures. Four serotypes of dengue virus (DENV1, DENV2, DENV3, and DENV4) have been isolated, all of which are pathogenic. Typical clinical manifestations of dengue fever are rapid onset, high fever, headache, severe pain in muscles, bones and joints, rash, bleeding tendency, swollen lymph nodes, decreased white blood cell count, and thrombocytopenia in some patients. Dengue virus infection often causes dengue classical fever (DF) and dengue hemorrhagic fever / shock syndrome (DHF / DSS). DF is a self-limiting febrile disease; while DHF / DSS is a severe disease that threatens the patient's life, and its main feature is a significant increase in vascular permeability, leading to plasma leakage. Every year, approximately 500,000 DHF / DSS patients require prompt hospitalization, with a mortality rate as high as 50%.

[0003] The disease is spread by Aedes mosquitoes (including Aedes aegypti and Aedes albopictus), so the epidemic is seasonal, and patients and latently infected persons are also the main sources of infection. In new epidemic areas, the disease mainly occurs in adults, while in local epidemic areas, the disease mainly occurs in children. The accelerated pace of globalization, the increase in personnel exchanges between different parts of the world, urbanization and the rapid growth of population have all contributed to the rapid spread of dengue fever.

[0004] After infection, dengue virus can proliferate in capillary endothelial cells and mononuclear phagocyte system and then enter blood circulation, forming the first toxemia. Then it will replicate in the mononuclear phagocyte system and lymphoid tissue and be released into the blood again, forming the second toxemia. Dengue virus forms immune complex with anti-dengue virus antibodies produced by the body, activates the complement system, and leads to increased vascular permeability. At the same time, the virus can inhibit the bone marrow, leading to leukopenia, thrombocytopenia and bleeding tendency.

[0005] Over the past 70 years, researchers have developed vaccines for DENV from various methods and angles, but there is still no vaccine or drug to prevent and treat DENV infection. Whether it is live attenuated vaccines, multivalent vaccines, nucleic acid vaccines, or protein component vaccines, they have encountered obstacles such as antigenic variation of DENV epidemic strains and antibody-dependent enhancement (ADE), and progress has been slow. Based on the significant increase in the number of DHF / DSS patients in recent years and the above research status, passive immunity or therapeutic monoclonal antibodies have once again attracted the attention of researchers. As a passive immunization strategy with mature technology, strong specificity, and good therapeutic effect, monoclonal antibodies have gradually become a hot topic of research. Summary of the invention

[0006] The purpose of the present invention is to provide a monoclonal antibody against four serotypes of dengue virus and its application. The monoclonal antibody of the present invention has strong binding ability with four serotypes of DENV virus, can detect four serotypes of dengue virus at the same time, and the antibody titer can reach 1:512000 or more, which has great application value.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a monoclonal antibody against four serotypes of dengue virus, wherein the amino acid sequence of the heavy chain of the monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of the light chain is shown as SEQ ID NO.2.

[0009] The present invention also provides an application of the monoclonal antibody against four serotypes of dengue viruses in the preparation of a drug for treating dengue fever.

[0010] The present invention also provides an application of the monoclonal antibody against four serotypes of dengue viruses in preparing a reagent for detecting or diagnosing dengue fever.

[0011] The present invention provides a monoclonal antibody against four serotypes of dengue virus and its application. The monoclonal antibody of the present invention has the ability to detect four serotypes of dengue virus at the same time, and the antibody dilution ratios of WB and ELISA are both within the appropriate range; the titer of the monoclonal antibody against four serotypes of DENV virus of the present invention can reach 1:512000 or more, has strong binding ability with four serotypes of DENV virus, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The results of the antibody titer test in mouse serum after four rounds of immunization in Example 1 are shown.

[0013] Figure 2This is a diagram of the monoclonal antibody subtype identification results in Example 2.

[0014] Figure 3 This is a graph showing the results of the monoclonal antibody titer determination in Example 2.

[0015] Figure 4 The figure is the result of the purity identification of the monoclonal antibody in Example 2, wherein M is the protein relative molecular mass standard, lanes 1 to 2 are the flow-through liquid, and lanes 3 to 6 are the monoclonal antibodies.

[0016] Figure 5 This is a diagram of the specific identification results of the monoclonal antibody in Example 2, wherein lane 1 is type 1 (Domain III); lane 2 is type 2 (Domain III); lane 3 is type 3 (Domain III); lane 4 is type 4 (Domain III); lane 5 is DENV1; lane 6 is DENV2; lane 7 is DENV3; and lane 8 is DENV4. DETAILED DESCRIPTION

[0017] The present invention provides a monoclonal antibody against four serotypes of dengue virus, wherein the amino acid sequence of the heavy chain of the monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of the light chain is shown as SEQ ID NO.2.

[0018] In the present invention, the amino acid sequence of SEQ ID NO.1 is:

[0019] GAEVVRPGVSVKISCKISGYKFTDYTMHWVKESHAKSLEWIGVISPF YGDATYSQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCARRGVTT RDWFAYWGQGTLVTVSAAKTTPKLVYPLGP.

[0020] In the present invention, the amino acid sequence of SEQ ID NO.2 is:

[0021] QVFVFVFLWLSGVDGDIVMTPSHKFMSTSVGDRVTITCKASQDVTTA VAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTYFTFTISNVQAED LAFYYCQQHYGAPFTFGSGTKLEIKRADAAPTGIQ.

[0022] The present invention also provides an application of the monoclonal antibody against four serotypes of dengue viruses in the preparation of a drug for treating dengue fever.

[0023] The present invention also provides an application of the monoclonal antibody against four serotypes of dengue viruses in preparing a reagent for detecting or diagnosing dengue fever.

[0024] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention. If no specific conditions are specified in the embodiments, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0025] Example 1

[0026] This example prepared monoclonal antibodies against four serotypes of dengue virus, and the specific operations were as follows:

[0027] (1) Material preparation:

[0028] DENV1, DENV2, DENV3, and DENV4 viruses were obtained from the Center for Disease Control and Prevention of the Southern Theater Command of the Chinese People’s Liberation Army;

[0029] Dengue virus (DENV) (type 1-4, strain New Guinea C / PUO-218 hybrid) E / Envelope Protein (Domain III, His Tag) was purchased from Beijing Sino Biological Technology Co., Ltd. (China);

[0030] BALB / cBALB / c mice were purchased from the Animal Experiment Center of Army Medical University;

[0031] KM mice were purchased from the Animal Experiment Center of Army Medical University;

[0032] Sp2 / 0 cells were donated by the Department of Clinical Biochemistry, Department of Laboratory Medicine, Army Medical University;

[0033] Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.;

[0034] HRP-labeled goat anti-mouse immunoglobulin G was purchased from Beijing Solebao Technology Co., Ltd.;

[0035] Dulbecco's minimal messenger medium (DMEM) high glucose, fetal bovine serum (FBS), 50× hypoxanthine thymine (HT), and 50× hypoxanthine aminotrexate thymine (HAT) were purchased from Gbico.

[0036] Polyethylene glycol PEG2000 (PEG2000) was purchased from Solarbio;

[0037] The 100 μm cell strainer was purchased from BD;

[0038] Penicillin / streptomycin dual antibody solution, red blood cell lysis buffer, and Tween-20 were purchased from Beyotime Biotechnology Co., Ltd.

[0039] Bovine serum albumin (BSA) was purchased from Saiguo Biotechnology Co., Ltd.;

[0040] Subtype identification antibodies (anti-IgG antibody, anti-IgG1 antibody, anti-IgG2a antibody, anti-IgG2b antibody, anti-IgG2c antibody, and anti-IgG3 antibody) were purchased from Beijing Sino Biological Technology Co., Ltd. (China);

[0041] Protein A+G affinity chromatography column and filler were purchased from Beyotime;

[0042] The BCA protein concentration determination kit was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0043] (2) Mouse immunization and serum antibody titer detection

[0044] DENV1, DENV2, DENV3, and DENV4 viruses were mixed at a protein mass ratio of 1:1:1:1, inactivated in a water bath at 56°C for 30 minutes, and then completely mixed with an equal volume of TH-Z93 adjuvant, and four rounds of immunization were performed on 6-8 week old female BALB / c mice. The first three rounds were subcutaneously injected at the back of the neck, with an injection volume of 100 μg / mouse, once every two weeks; after three immunizations, the mouse orbital blood was collected, and the mouse serum antibody titer was measured to be above 1:10000, and then the mouse was intraperitoneally injected at 200 μg / mouse to strengthen the immunity.

[0045] After the four rounds of immunization, the orbital blood of mice was collected for testing, and the serum antibody titer was tested with DENV1, DENV2, DENV3, and DENV4 viruses. The orbital serum of mice before immunization was used as a negative control, and the OD 450 ≈1.0, P / N>2.1 (P and N represent the OD measured after the positive serum and negative serum were removed from the blank, respectively 450 ) as the positive critical value of the test result. The test results of mouse serum antibody titer are as follows Figure 1 shown.

[0046] from Figure 1It can be seen that after four rounds of immunization, the final mouse serum antibody titer can reach above 1:512000, and cell fusion can be performed.

[0047] (3) Preparation of mouse peritoneal macrophages (feeder cells)

[0048] One day before cell fusion, KM mice were killed by dislocating the neck and then immersed in 75% alcohol for disinfection for 5 minutes; the mouse limbs were fixed, the peritoneum was cut open with forceps, and 10mL HAT (purchased from Gbico) was drawn with a syringe and injected into the mouse peritoneal cavity. After gently kneading the abdomen for 1 minute, 8mL of culture medium containing peritoneal macrophages was aspirated. 50mL of HAT culture medium (DMEM containing 20% ​​FBS, 1% penicillin / streptomycin double antibody solution, 2% HAT) was added to the aspirated culture medium containing peritoneal macrophages, mixed and dispensed into 96-well cell culture plates at 100μL / well and placed at 37°C and 5% CO 2 Incubate overnight in an incubator.

[0049] (4) Recovery and expansion of myeloma cells (Sp2 / 0) before cell fusion

[0050] Take out the frozen Sp2 / 0 cells from the liquid nitrogen tank, thaw quickly at 42°C, and immediately add them to a 5mL centrifuge tube containing complete culture medium (DMEM containing 20% ​​FBS and 1% penicillin / streptomycin double antibody solution). Centrifuge the centrifuge tube at 1000rpm for 5min, discard the supernatant, blow the cell pellet evenly with 10mL complete culture medium, and place it at 37°C and 5% CO. 2 Sp2 / 0 cells with good condition and fast growth rate were selected for cell fusion experiment.

[0051] (5) Cell fusion

[0052] The spleen of mice that have completed four rounds of immunization was taken and fully ground to collect spleen cells. After counting the cells, 2×10 6 splenocytes and 4×10 5 Mix Sp2 / 0 cells in logarithmic growth phase, centrifuge at 1000rpm for 10min, and discard the supernatant. Place the centrifuge tube in 37℃ warm water, slowly add 1mL preheated PEG2000 within 45s, mix well and let stand for 1min. After standing, add the stop solution DMEM according to the following procedure: add 2mL DMEM in the first 30s, add 8mL DMEM in the middle 30s, and add 20mL DMEM in the last 30s.

[0053] The centrifuge tube was centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. The cells were resuspended in HAT medium and added to the 96-well cell culture plate containing feeder cells prepared in (3) at a rate of 100 μL / well. After 5 days of culture, half of the HAT medium was replaced with fresh medium, and after 15 days, it was replaced with HT medium.

[0054] (6) Positive hybridoma cell screening and subcloning

[0055] When a large cell clone appeared in the cell culture plate, it was coated with four different serotypes of DENV virus and the positive clone wells were screened by indirect ELISA. A total of 18 clone wells with high positive values ​​were screened, and the cells in the clone wells were expanded and cultured. When the cells grew to 70%, they were subcloned by limiting dilution. After three subclones, a positive hybridoma cell line that can stably secrete monoclonal antibodies was obtained.

[0056] (7) Preparation and purification of monoclonal antibodies

[0057] Nine-week-old female BALB / c mice were intraperitoneally injected with 300 μL of Freund's incomplete adjuvant per mouse. Four days later, 2×10 positive hybridoma cells in the logarithmic growth phase (6) were obtained. 6 The cells were intraperitoneally injected into the above BALB / c mice. After 8 days, the ascites was collected. The red blood cells were removed after centrifugation at 3000 rpm for 10 min. The supernatant was aspirated after centrifugation at 8000 rpm for 5 min. The supernatant was purified by ascites purification through a Protein A+G affinity chromatography column to obtain a monoclonal antibody named 8-9D.

[0058] Example 2

[0059] The monoclonal antibody prepared in Example 1 was subjected to subtype identification, purity and concentration identification, titer determination, and specificity identification.

[0060] (1) Subtype identification

[0061] Anti-IgG antibody, anti-IgG1 antibody, anti-IgG2a antibody, anti-IgG2b antibody, anti-IgG2c antibody, and anti-IgG3 antibody were used as secondary antibodies to identify antibody subtypes by indirect ELISA. The results were as follows: Figure 2 shown.

[0062] from Figure 2 It can be seen that the monoclonal antibody prepared in Example 1 is of IgG1 type.

[0063] (2) Potency determination

[0064] DENV1, DENV2, DENV3, and DENV4 viruses were used to test the titers of the multiple monoclonal antibodies prepared in Example 1. PBS solution was used as a negative control and the OD 450≈1.0, P / N>2.1 (P and N represent the OD measured after the positive antibody and negative control were removed from the blank, respectively 450 ) as the positive critical value of the test result. Figure 3 As shown, from Figure 3 It can be seen that the titer of the monoclonal antibody prepared in Example 1 reaches 1:512000 or more.

[0065] (3) Purity and concentration identification

[0066] The flow-through from the preparation of 8-9D monoclonal antibody in Example 1 and the purification process was mixed with 5×SDS protein loading buffer at a ratio of 1:4, boiled for 5 minutes until the protein was denatured, and subjected to SDS-PAGE electrophoresis for purity identification. The results were as follows: Figure 4 As shown, M is the protein relative molecular mass standard, lanes 1 to 2 are flow-throughs, and lanes 3 to 6 are monoclonal antibodies.

[0067] from Figure 4 It can be seen that there are two bands in the lanes of monoclonal antibodies, namely: 25KDa light chain and 55KDa heavy chain; the flow-through contains many impurity protein bands, while the monoclonal antibodies have no obvious impurity proteins, indicating that the purified monoclonal antibodies have a high purity.

[0068] The concentration of the monoclonal antibody prepared in Example 1 was measured using a BCA protein concentration quantitative determination kit, and the concentration detection result of the monoclonal antibody was: 4.15 mg / mL.

[0069] (4) Specific identification

[0070] The specificity of the monoclonal antibody prepared in Example 1 was identified by Western blot.

[0071] Take DENV1, DENV2, DENV3, DENV4 virus liquid and commercially available Denguevirus (DENV) (type1-4) E / EnvelopeProtein (DomainIII, HisTag), perform SDS-PAGE electrophoresis, transfer to membrane, block, and wash with TBST. Take 8-9D monoclonal antibody, dilute it at the ratio of antibody: 5% BSA = 1:1000 as the primary antibody, and incubate it at 4°C overnight; the next day after washing with TBST, add HRP-labeled goat anti-mouse immunoglobulin G diluted at the ratio of antibody: 5% BSA = 1:5000 as the secondary antibody for incubation. After incubation for 60 minutes, wash with TBST, and observe the antibody binding effect with ECL color development. The results are as follows Figure 5As shown, lane 1 is type 1 (Domain III); lane 2 is type 2 (Domain III); lane 3 is type 3 (Domain III); lane 4 is type 4 (Domain III); lane 5 is DENV1; lane 6 is DENV2; lane 7 is DENV3; and lane 8 is DENV4.

[0072] from Figure 5 It can be seen that at a dilution ratio of 1:1000, the monoclonal antibody prepared in Example 1 can specifically bind to and recognize type 1-4 (Domain III) and DENV1, DENV2, DENV3, and DENV4 virus fluids.

[0073] The monoclonal antibody was sequenced and the sequence of the antibody is as follows:

[0074] Amino acid sequence of the heavy chain (SEQ ID NO.1):

[0075] GAEVVRPGVSVKISCKISGYKFTDYTMHWVKESHAKSLEWIGVISPFYGDATYSQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCARRGVTTTRDWFAYWGQGTLVTVSAAKTTPKLVYPLGP

[0076] Amino acid sequence of light chain (SEQ ID NO.2):

[0077] QVFVFVFLWLSGVDGDIVMTPSHKFMSTSVGDRVTITCKASQDVTTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTYFTFTISNVQAEDLAFYYCQQHYGAPFTFGSGTKLEIKRADAAPTGIQ

[0078] In summary, the monoclonal antibodies against four serotypes of dengue virus of the present invention have high titers, have strong binding abilities to four serotypes of DENV viruses, and can realize simultaneous detection of four serotypes of dengue virus.

[0079] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A monoclonal antibody against four serotypes of dengue virus, It is characterized in that The amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain is shown in SEQ ID NO.

2.

2. Use of the monoclonal antibody against four serotypes of dengue virus according to claim 1 in the preparation of a drug for treating dengue fever.

3. Use of the monoclonal antibody against four serotypes of dengue virus according to claim 1 in the preparation of a reagent for detecting or diagnosing dengue fever.

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